ENVIRONMENT AND AUTOIMMUNITY

Behavioral abnormalities in female mice followingadministration of aluminum adjuvantsand the human papillomavirus (HPV) vaccineGardasil

Rotem Inbar1,2 • Ronen Weiss3,4 • Lucija Tomljenovic1,5 •

Maria-Teresa Arango1,6 • Yael Deri1 • Christopher A. Shaw5•

Joab Chapman1,7 • Miri Blank1 • Yehuda Shoenfeld1,8

� Springer Science+Business Media New York 2016

Abstract Vaccine adjuvants and vaccines may induce autoimmune and inflammatory manifestations in susceptible

individuals. To date most human vaccine trials utilize aluminum (Al) adjuvants as placebos despite much evidence

showing that Al in vaccine-relevant exposures can be toxic to humans and animals. We sought to evaluate the effects of Al

adjuvant and the HPV vaccine Gardasil versus the true placebo on behavioral and inflammatory parameters in female mice.

Six-week-old C57BL/6 female mice were injected with either, Gardasil, Gardasil ? pertussis toxin (Pt), Al hydroxide, or,

vehicle control in amounts equivalent to human exposure. At 7.5 months of age, Gardasil and Al-injected mice spent

significantly more time floating in the forced swimming test (FST) in comparison with vehicle-injected mice (Al,

p = 0.009; Gardasil, p = 0.025; Gardasil ? Pt, p = 0.005). The increase in floating time was already highly significant at

4.5 months of age for the Gardasil and Gardasil ? Pt group (p B 0.0001). No significant differences were observed in the

number of stairs climbed in the staircase test which measures locomotor activity. These results indicate that differences

observed in the FST were unlikely due to locomotor dysfunction, but rather due to depression. Moreover, anti-HPV

antibodies from the sera of Gardasil and Gardasil ? Pt-injected mice showed cross-reactivity with the mouse brain protein

extract. Immunohistochemistry analysis revealed microglial activation in the CA1 area of the hippocampus of Gardasil-

injected mice. It appears that Gardasil via its Al adjuvant and HPV antigens has the ability to trigger neuroinflammation

and autoimmune reactions, further leading to behavioral changes.

Keywords Gardasil � Aluminum � ASIA syndrome � Autoantibodies � Autoimmunity � Neuroinflammation

Abbreviations

Al Aluminum

ASIA Autoimmune/autoinflammatory syndrome

induced by adjuvants

b2-GPI b2-Glycoprotein I

FST Forced swimming test

HPV Human papilloma virus

Pt Pertussis toxin

U. S FDA United States Food and Drug Administration

& Yehuda Shoenfeld

shoenfel@post.tau.ac.il

1 Zabludowicz Center for Autoimmune Diseases, Sheba

Medical Center, Tel Hashomer, Sackler Faculty of Medicine,

Tel-Aviv University, 52621 Israel, Israel

2 Department of Obstetrics and Gynecology, Sheba Medical

Center, 52621 Tel Hashomer, Israel

3 Sagol School of Neuroscience, Tel Aviv University,

69978 Ramat Aviv, Israel

4 Department of Physiology and Pharmacology, Sackler

Faculty of Medicine, Tel Aviv University,

69978 Ramat Aviv, Israel

5 Neural Dynamics Research Group, Department of

Ophthalmology and Visual Sciences, University of British

Columbia, 828 W. 10th Ave, Vancouver, BC V5Z 1L8,

Canada

6 Doctoral Program in Biomedical Sciences, Universidad del

Rosario, Bogota 111221, Colombia

7 Department of Neurology and Sagol Neuroscience Center,

The Sheba Medical Center, 52621 Tel Hashomer, Israel

8 Incumbent of the Laura Schwarz-Kip Chair for Research of

Autoimmune Diseases, Sackler Faculty of Medicine,

Tel-Aviv University, 69978 Ramat Aviv, Israel

Yehuda Shoenfeld

123

Immunol Res

DOI 10.1007/s12026-016-8826-6

Introduction

Like other drugs, vaccines can cause adverse events, but

unlike conventional medicines, which are prescribed to

people who are ill, vaccines are administered to healthy

individuals. Hence, there is an added concern regarding

risks associated with vaccinations. While most reported

side effects from vaccines are mild and transient, serious

adverse events do occur and can even be fatal [1, 2].

There are currently major stumbling blocks in our

understanding of the exact mechanisms by which such

events can be triggered. The main reason for this is the

poor methodological quality of many clinical studies that

evaluate vaccine safety and the lack of in-depth research

into adverse phenomena [3]. In addition, adverse events

may not fit into a well-defined category of an autoimmune

disease but rather, present themselves as a constellation of

non-specific symptoms (i.e., arthralgia, myalgia, fatigue,

nausea, weakness, paresthesia, depression, mild cognitive

disturbances) [2]. Another complicating factor in

researching vaccine-related adverse events is that the

latency period between vaccination and the development of

an overt and diagnosable autoimmune and/or neurological

disease can range from days to many months [4–6], likely

depending on individuals’ genetic predispositions and other

susceptibility factors (i.e., previous history of autoimmune

disease or previous history of adverse reactions to

vaccines).

From the above, it is clear that establishing a definite

causal link between vaccinations and disease manifesta-

tions in humans remains a complex task. Thus, the poten-

tial risks from vaccines remain currently ill-understood and

controversial. A further obfuscation to our understanding

of potential risks from vaccinations stems from the per-

sistent use of aluminum (Al) adjuvants-containing placebos

in vaccine trials [7]. Indeed, contrary to popular assump-

tions of inherent safety of Al in vaccines, there is now

compelling data from both human and animal studies

which implicates this most widely used adjuvant in the

pathogenesis of disabling neuroimmuno-inflammatory

conditions [8–11].

Due to their capability of enhancing the immune

response to foreign antigens, substances with adjuvant

properties have been used for decades to enhance the

immunogenicity of human and animal vaccines [12].

Because of their immune-potentiating capacity, adjuvants

enable the usage of smaller amount of antigens in vaccine

preparations and are thus attractive from a commercial

standpoint. Nonetheless, enhanced immunogenicity also

implies enhanced reactogenicity. Indeed, although Al acts

as an effective vehicle for the presentation of antigens, this

process is not always benign since the adjuvant itself is

intrinsically capable of stimulating pathological immune

and neuro-inflammatory responses [9–11, 13–16]. In spite

of these data, it is currently maintained by both the phar-

maceutical industry and drug-regulating agencies that the

concentrations at which Al is used in vaccines does not

represent a health hazard [17].

Apart from potential hazards associated with adjuvant

use, other ingredients in vaccines also have the capacity of

provoking undesirable adverse events. Indeed, since the

mechanisms by which the host’s immune system responds

to vaccination resemble the ones involved in the response

to infectious agents, a recombinant or a live attenuated

infectious antigen used for vaccination, may inflict a range

of immune and autoimmune responses similar to its par-

allel infectious agent [18, 19].

The HPV vaccine Gardasil is one of many vaccines

currently on the market that is adjuvanted with Al. Since

the licensure by the US Food and Drug Administration

(FDA) and subsequent introduction on the market in 2009,

the HPV vaccine has been linked to a variety of serious

neurological and autoimmune manifestations. Notably, out

of 152 total cases identified via PubMed 129 (85 %) are

related to neuro-ophthalmologic disorders (Table 1). It

should be noted that the pattern of adverse manifestations

emerging from HPV vaccine case reports, matches that

reported through various vaccine safety surveillance sys-

tems worldwide, with nervous system and autoimmune

disorders being the most frequently reported [20].

Like most other vaccine safety trials, the trials for the

HPV Gardasil vaccine utilized an Al-containing placebo

[21, 22] and hence the safety profile of the vaccine remains

obscured by the use of a potentially toxic placebo [7].

Thus, in order to investigate better, the safety profile of

Gardasil, as well as the Al adjuvant, in the current study,

we evaluated and compared the effects of Al and whole

HPV vaccine formulation versus that of a true placebo on

behavioral, neurohistological and autoimmune parameters

in young female C57BL/6 mice.

Materials and methods

Mice husbandry

Six-week-old C57BL/6 female mice were obtained from

Harlan Laboratories (Jerusalem, Israel) and were housed in

the animal facility at Sheba Medical Center. The mice were

raised under standard conditions, 23 ± 1 �C, 12-light cycle(6:30 am–6:30 pm) with ad libitum access to food and

water. The Sheba Medical Center Animal Welfare Com-

mittee approved all procedures.

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Table 1 Summary of cases of autoimmune and inflammatory manifestations following HPV vaccination reported in the peer-reviewed medical

literature

Number of

case reports

Age Symptoms/main clinical features Final diagnosis References

2 17 Visual impairments ADEM [52]

20 Headache, nausea, vomiting, diplopia [53]

5 16 Upper limb pseudoathetosis CIS/MS/ [54]

16 Acute hemiparesis Clinically definite MS

21 Incomplete TM, left optic neuritis

25 Headache, incomplete TM

26 Incomplete TM, brainstem syndrome

2 19 Leg numbness, mid-thoracic back pain Demyelinating disease

unspecified

[55]

18 Blurriness, paresthesia, optic neuritis

1 11 Mood swings, abnormal eye movements, dizziness, leg weakness,

myoclonic jerks

Opsoclonus myoclonus [56]

4 17 Back pain, progressing spastic paraparesis, right arm weakness, left eye

visual loss

Neuromyelitis optica [57]

14 Back pain, right thigh dysesthesias, left optic neuritis

13 TM with flaccid paraplegia

18 Back pain and leg weakness, complete loss of monocular vision

2 16 Visual loss, headaches, left hemiparesis Optic neuritis [58]

17 Visual disturbances, demyelinating lesions [59]

2 27 Paresthesia, demyelinating lesions TM fitting the criteria for MS [59]

26 Progressive paresthesia, demyelinating lesions

1 15 Facial paralysis Bell’s palsy [59]

1 12 Nausea, vertigo, severe limb and truncal ataxia, and persistent nystagmus Cerebellar ataxia [60]

1 19 Chronic (3 months) disabling shoulder pain Brachial neuritis [61]

53 12–39 Orthostatic intolerance, severe non-migraine-like headache, excessive

fatigue, cognitive dysfunction, gastrointestinal discomfort, widespread

neuropathic pain

Dysautonomia, POTS,

orthostatic intolerance and

CRPS

[62]

40 11–17 Headaches, general fatigue, coldness of the legs, limb pain and weakness,

orthostatic intolerance, tremors, persistent asthenia

[63]

6 20 Weight loss, dizziness, fatigue, exercise intolerance [64]

22 Diarrhea, weight loss, fatigue, dizziness, syncope

12 Syncope, pre-syncope, dizziness, small fiber neuropathy

15 Dizziness, headache, pre-syncope, syncope

Paresthesia, tachycardia, fatigue, headache,

14 diarrhea, weight loss

18 Paresthesia, leg pain, orthostatic intolerance, Fatigue, dizziness

4 16 Paresthesia, numbness, limb paralysis, pain [65]

13 Allodynia, numbness, severe pain

15 Paresthesia, numbness, severe pain

12 Paresthesia, muscle weakness, pain

1 14 Headaches, dizziness, recurrent syncope, orthostatic intolerance, fatigue,

myalgias, tachycardia, dyspnea, visual disturbances, phonophobia,

cognitive impairment, insomnia, gastrointestinal disturbances, weight

loss

[66]

2 11 Widespread neuropathic pain, paresthesia, insomnia, profound fatigue Fibromyalgia [67]

14 Widespread neuropathic pain and paresthesia

1 32 Paresthesia, muscle twitching, myalgia, fatigue, hyperhidrosis, and

tachycardia, exercise intolerance

Autoimmune myotonia [68]

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Table 1 continued

Number of

case reports

Age Symptoms/main clinical features Final diagnosis References

3 14 Skin rash, fever, nausea, stomach aches, headache, insomnia, night sweats,

arthralgia, anxiety, depression, amenorrhea, elevated serum levels of

follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and

low levels of estradiol

POF [69]

13 Depression, sleep disturbance, light-headedness, tremulousness, anxiety,

cognitive dysfunction, amenorrhea, high serum levels of FSH and LH

with undetectable estradiol

[70]

21 A menorrhea preceded by oligomenorrhea, high serum levels of FSH and

LH and low estradiol

3 16 5 months amenorrhea preceded by 12 months oligomenorrhea, hot flashes,

low serum levels of estradiol and Anti-Mullerian hormone

18 6 months amenorrhea, low serum levels of estradiol and Anti-Mullerian

hormone

15 3 months amenorrhea preceded by 9 months oligomenorrhea, hot flashes,

low serum levels of estradiol and undetectable Anti-Mullerian hormone

2 15 Vasculitic rash, soft tissue swellings of Vasculitis [71]

ankles and forearms, arthralgia, lethargy, epistaxis

15 Severe flare of cutaneous vasculitis

1 16 Fatigue associated with prolonged menorrhagia, antiplatelet autoantibodies Thrombocytopenic purpura [72]

1 11 Jaundice, hepatosplenomegaly elevated serum aminotransferases Autoimmune hepatitis [73]

1 26 Severe constant epigastric pain, vomiting, fever Pancreatitis [74]

3 17 Arthralgias, pruritic rashes on lower extremities, bipedal edema, livedo

reticularis, proteinuria, positive ANA and anti-dsDNA antibodies

SLE [75]

45 Intermittent fever, generalized weakness, oral ulcers, alopecia, malar rash,

photosensitivity, arthritis, intestinal pseudo-obstruction, ascites, positive

ANA, anti-dsDNA, anti-Ro/SSA and anti- La/SSB antibodies

58 Malar and scalp rashes, fever, easy fatigability, cervical lymph nodes, gross

hematuria and pallor, severe anemia and thrombocytopenia, active

nephritis, patient expired a day after hospital admission

6 32 Fatigue, severe myalgia, polyarthralgia, anorexia, severe skin rash, malar

rash, aphtous stomatitis, pharyngodynia, cervical lymphadenopathy,

alopecia, severe weight loss, anemia, positive ANA and anti-dsDNA

antibodies

[76]

29 Weakness, diarrhea, malar rash, photosensitivity, arthritis, alopecia, severe

weight loss, proteinuria, positive ANA and anti-dsDNA antibodies

16 High-grade fever, generalized asthenia, diffuse polyarthralgia, multiple

erythematous annular cutaneous lesions on the face, trunk, and lower

limbs, positivie ANA and lupus anticoagulant

16 Fever, pharyngodynia, erythematous skin lesions of elbows and knees,

generalized asthenia, anorexia, polyarthralgia, anti-cardiolipin and lupus

anticoagulant

19 Mild arthralgia, dyspnea, cervical lymphadenopathy, skin rash, positive

ANA and anti-dsDNA antibodies

13 Erythematous facial rash, fever, periorbital edema, weight loss, malaise,

fatigue, alopecia, cervical, axillary and inguinal lymphadenopathy,

anemia, thrombocytopenia, positive ANA, anti-RNP, anti-Smith and

anti-RO/SSA antibodies

1 19 Myalgia, arthralgia, generalized weakness, oral ulcers, Raynaud’s

phenomenon, alopecia, headache, dyspnea, tachycardia, positive ANA,

anti-Sm, anti-Ro, anti-RNP, anti-dsDNA, leuko-penia, and complement

consumption

[77]

1 20 Myalgias, arthral-gias, livedo reticularis, Raynaud’s phenomenon,

headache, tinnitus, positive ANA, lupus anticoagulant and anti-CCP

Rheumatoid arthritis [77]

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Injection procedures and experimental design

Six-week-old C57BL/6 female mice received three injec-

tions (spaced 1 day apart) of either (a) quadrivalent HPV

vaccine Gardasil, (b) Gardasil ? pertussis toxin (Pt), (c) Al

hydroxide or (d) vehicle control (19.12 mg/mL NaCl,

1.56 mg/mL L-histidine). The number of injected animals

was 19 per experimental group. Gardasil, Al and vehicle

were injected intramuscularly (i.m.), while the Pt was given

intraperitoneally (ip). The amount of injected Al and the

HPV vaccine was the equivalent of human exposure. In

particular, each mouse in the Gardasil and Gardasil ? Pt

group received 0.25 ll of Gardasil (dissolved in 20 ll ofvehicle solution). 0.25 ll of Gardasil is the equivalent of ahuman dose since the average weight of a six-week-old

mice is approximately 20 g. Gardasil is given as a 0.5-mL

dose to teenage girls of cca 40 kg. Thus, a 20-g mouse

receives cca 2000 9 less of the vaccine suspension than a

human. Similarly, each mouse in the Al adjuvant group

received 5.6 lg/kg body weights Al hydroxide dissolved in

20 ll vehicle solution. A single Gardasil dose contains

225 lg of Al and is given to a cca 40-kg female. This

equates to 5.6 lg Al hydroxide/kg body weight. The mice

in the Pt group received 250 ng of Pt with each injection of

Gardasil. Pt was added to this group for the purpose of

damaging the blood–brain barrier. Since the actual adju-

vant form used in Gardasil, amorphous Al hydroxyphos-

phate sulfate (AAHS), is a proprietary brand of the vaccine

manufacturer and is not commercially available, we used

Alhydrogel as a substitute.

Five out of 19 animals from each of the four experi-

mental groups were used for sera collection purposes.

These animals were not subjected to behavioral testing as

sera were collected via retro-orbital bleeding which is a

stressful procedure that in addition often leads to vision

deficits. The behavior of mice was evaluated at three and

6 months post-immunization for (1) locomotor function

and depression by the forced swimming test (FST), (2)

locomotor and explorative activity by the staircase test and

(3) cognitive functions by the novel object recognition test.

Following the first round of behavioral testing at

4.5 months of age, five mice from each of the four

experimental groups were killed and brain tissues were

collected and processed for histological examinations.

Blood specimens were also collected at this time for

serological analysis.

Behavioral tests

Forced swimming test

The FST is the most widely used model of depression in

rodents. It is commonly used for evaluation of antide-

pressant drugs, and experiments aimed at inducing and

examining depressive-like states in basic and pre-clinical

research [23, 24]. Nonetheless, it should be noted that

increased floating time in the FST apart from being

indicative of depressive behavior can also indicate loco-

motor dysfunction. For the purpose of this test, mice were

placed in individual glass beakers (height 39 cm, diameter

21.7 cm) with water 15 cm deep at 25 �C. On the first day,

mice were placed in the cylinder for a pretest session of

10 min, and later were removed from the cylinder, and then

returned to their home cages. Twenty-four hours later (day

2), the mice were subjected to a test session for 6 min. The

behavioral measure scored was the duration (in seconds) of

immobility or floating, defined as the absence of escape-

oriented behaviors, such as swimming, jumping, rearing,

sniffing or diving, recorded during the 6-min test.

Staircase test

Locomotor, explorative activity and anxiety were evalu-

ated by the staircase test, as described previously by Kat-

zav et al. [25]. In this test, stair-climbing and rearing

frequency are recorded as measures of general locomotor

function, exploratory activity and anxiety/attention. The

staircase maze consisted of a polyvinyl chloride enclosure

with five identical steps, 2.5 9 10 9 7.5 cm. The inner

height of the walls was constant (12.5 cm) along the whole

length of the staircase. The box was placed in a room with

constant lighting and isolated from external noise. Each

Table 1 continued

Number of

case reports

Age Symptoms/main clinical features Final diagnosis References

1 16 Knee joint swelling, low back, buttock and chest wall pain, elevated

leukocyte count in the synovial fluid, elevated C-reactive protein

Juvenile spondyloarthropathy [77]

Out of 152 reported cases, 129 (85 %) relate to neuro-ophthalmic disorders

ANA antinuclear antibodies; ADEM acute disseminated encephalomyelitis; CIS clinically isolated syndrome; CRPS complex regional pain

syndrome; MS multiple sclerosis; POF primary ovarian failure; POTS postural orthostatic tachycardia syndrome (disorder of the autonomic

nervous system); SLE systemic lupus erythematosus; TM transverse myelitis

Environment and Autoimmunity

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mouse was tested individually. The animal was placed on

the floor of the staircase with its back to the staircase. The

number of stairs climbed and the number of rears were

recorded during a 3-min period. Climbing was defined as

each stair on which the mouse placed all four paws; rearing

was defined as each instance the mouse rose on hind legs

(to sniff the air), either on the stair or against the wall. The

number of stairs descended was not taken into account.

Before each test, the animal was removed and the box

cleaned with a diluted alcohol solution to eliminate smells.

Novel object recognition test

This is a visual recognition memory test based on a method

described by Tordera et al. [24]. The apparatus, an open-

field box (50 9 50 9 20 cm), was constructed from ply-

wood painted white. Three phases (habituation, training

and retention) were conducted on three separate test days.

Before the training session, the mice were individually

habituated by allowing them to explore the box for 10 min

(day 1). No data were collected at this phase. During

training sessions (day 2), two identical objects were placed

into the box in the northwest and southeast corners (ap-

proximately 5 cm from the walls), 20 cm away from each

other (symmetrically) and then the individual animal was

allowed to explore them for 5 min. Exploration of an

object was defined as directing the nose to the object at a

distance of B1 cm and/or touching it with the nose and

rearing at the object; turning around or sitting near the

object was not considered as exploratory behavior. The

time spent in exploring each object was recorded as well as

the number of interactions with both objects. The animals

were returned to their home cages immediately after

training. During the retention test (day 3), one of the

familiar objects used during the training session was

replaced by a novel object. Then, the animals were placed

back into the box and allowed to explore the objects for

5 min. The same parameters were measured as during the

training session, namely the time spent in exploring each of

the two objects and the number of interactions with them.

All objects were balanced in terms of physical complexity

and were emotionally neutral. The box and the objects

were thoroughly cleaned by 70 % alcohol before each

session to avoid possible instinctive odorant cues. A pref-

erence index, a ratio of the amount of time spent exploring

any one of the two items (old and new in the retention

session) over the total time spent exploring both objects,

was used to measure recognition memory.

Statistical analysis

Results are expressed as the mean ± SEM. The differences

in mean for average immobility time in the FST, the

staircase test parameters (number of rearing and stair-

climbing events) and novel object recognition were eval-

uated by ANOVA and Tuckey for multiple comparisons in

the post hoc analysis. Significant results were determined

as p\ 0.05.

Brain perfusion and fixation

The mice were anesthetized by an i.p. injection of ketamine

(100 mg/kg) and xylazine (20 mg/kg) and killed by tran-

scardiac perfusion with phosphate-buffered saline (PBS)

followed by perfusion with 4 % paraformaldehyde (PFA,

Sigma-Aldrich Israel Ltd., Rehovot Israel) in phosphate

buffer (PO4, pH 7.4). After perfusion, the brain was quickly

removed and fixed overnight in 4 % PFA (in PO4, pH 7.4)

at 4 �C. On the following day, the brain was cryoprotected

by immersion in 30 % sucrose in 0.1 M PO4 (pH 7.4) for

24–48 h at 4 �C before brain cutting. Frozen coronal

Sects. (30–50 lm) were cut on a sliding microtome (Leica

Microsystems GmbH, Wetzlar, Germany), collected seri-

ally and kept in a cryoprotectant at -20 �C until staining.

Detection of autoantibodies in the sera

The levels of autoantibodies in the mice sera were tested by

a homemade ELISA 1 month post-injection. Briefly,

ELISA plates (M9410, Sigma-Aldrich) were coated sepa-

rately with 20 lg/well of different antigens: Gardasil

which contains the HPV L1 major capsid protein of HPV

types 6, 11, 16 and 18, mouse brain protein extract, mouse

brain phospholipid extract, Al hydroxide, dsDNA and

b2glycoprotein-I (b2GPI). The plates were incubated

overnight at 4 �C, washed and blocked with 3 %BSA in

PBS 1 h at 37 �C. Sera were added at dilution of 1:200 for

2 h at room temperature. The binding was probed with goat

anti-mouse IgG conjugated to alkaline phosphatase at

concentration of 1:5000 for 1 h at 37C. Following appro-

priate substrate, the data were read by ELISA reader at

405 nm.

Inhibition assay

Brain protein extracts were prepared by lysis of brains from

five healthy C57BL/6 mice, using ice-cold lysis buffer

containing 50 mM Tris (pH 7.5), 150 mM NaCl, 10 %

glycerol, 1 % Triton X-100, 1 mM EDTA,1 mM PMSF,

1 mM sodium vanadate, 0.1 % protease inhibitor mixture

(Sigma-Aldrich L-4391 St Louis, MO, USA) for 30 min on

ice and centrifuged at 13,000 rpm for 20 min. The lysate

was dialyzed against PBS. Protein concentration was

determined by BCA Protein Assay Kit (Pierce, Thermo

scientific, Rockford, IL, USA).

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ELISA plates were coated with the HPV vaccine Gar-

dasil which contains the HPV L1 major capsid protein of

HPV types 6, 11, 16 and 18. Following blocking with 5 %

skim milk powder, sera from the immunized mice, at dif-

ferent dilutions 1:200–1:10,000, were added to the plates in

order to define 50 % binding of the sera to the HPV. Next,

dilutions of sera which showed 50 % binding to HPV were

incubated overnight at 4 �C with different concentrations

of mouse brain protein extract (10–50 lg/ml) as the inhi-

bitor. The following day, the mixtures were subjected to

ELISA plates coated with HPV for 2 h at room tempera-

ture. The binding of the antibodies which did not create

complex with the brain protein extract was probed with

anti-mouse IgG conjugated to alkaline phosphatase, fol-

lowed by the appropriate substrate. The percentage of

inhibition was calculated as follows: % inhibi-

tion = 100 - [(OD of tested sample without inhibi-

tor - OD of tested sample with inhibitor)/(OD of tested

sample without inhibitor)] 9 100.

Brain tissue immunostaining

Brain sections were stained free-floating, incubated with

the first antibodies overnight at 4 �C. The slices were then

washed in PBS ? 0.1 % Triton X-100 and incubated at

room temperature for 1 h with the corresponding fluores-

cent chromogens-conjugated secondary antibody. Sec-

tions were stained for specific antigens with antibodies

against activated microglia (anti-Iba-1, polyclonal, Abcam,

Cambridge, UK) and astrocytes (anti-GFAP monoclonal,

Dako, Carpinteria, CA, USA). Counter staining was per-

formed with Hoechst (Sigma-Aldrich Israel Ltd., Rehovot

Israel).

Image acquisition, quantification and statistical analyses

Iba-1 and GFAP immunostaining was visualized

using 9 4/0.1 NA, 9 10/0.25 NA and 9 40/0.65 NA

objective lenses on a Nikon eclipse 50i fluorescence

microscope equipped with a Nikon DS Fi1 camera. In order

to minimize bleaching of the fluorescence, images were

obtained by serially moving the slide with no fluorescence

and then acquiring the images in a standard manner. All

sections were then studied quantitatively for differences in

immunostaining density among the groups, using Image J

software (NIH, USA). Region of interests (ROIs) was

drawn manually using the ‘Polygon selection’ tool. Brain

regions were identified using a mouse brain atlas. ROIs

were chosen to represent anatomical regions previously

shown to be involved in cognition and/or to exhibit vari-

able sensitivity to neuroinflammation in other models. The

mean intensity of the specific ROIs (910 magnification)

was recorded for each individual animal recorded

(Analyze � Measure), and data were analyzed using SPSS

statistical software (version 15.0). Univariate analysis was

conducted for each ROI/Antibody separately using ‘group’

as a fixed factor and ‘experiment’ as a Covariate. Post hoc

analysis, one-way ANOVA, Student’s t test, simple

regression or correlation analysis was used when appro-

priate, according to the experimental design. Significance

level was determined in one-tailed and two-tailed tests. The

level of statistical significance of differences is p\ 0.05.

Results

Behavioral tests

The ANOVA analysis showed significant differences in the

performance of the mice in the forced swimming and the

staircase tests 3 months after injection (Fig. 1). The

specific differences were detected by the post hoc test

which showed that the two groups injected with the Gar-

dasil vaccine spent significantly more time floating com-

pared to control mice and Al-injected mice (Fig. 1a). No

significant differences were found between the groups in

the overall memory skills (measured by the novel object

recognition test), locomotor function, exploratory activity

and anxiety which were measured in the staircase apparatus

(Fig. 1c).

The analysis after the behavioral testing at 6 months

post-injection demonstrated that the alterations in the FST

performance were sustained in the group injected with

Gardasil ? Pt compared to control mice (p = 0.024;

Fig. 1b), indicating that the effect of Gardasil ? Pt expo-

sure was long-lasting. Moreover, at 6 months post-injec-

tion, the Al-injected group likewise spent significantly

more time floating compared to the control group

(p = 0.044, Fig. 1b). Although the Gardasil group showed

increased floating time compared to the vehicle-injected

control group, the observed difference was not statistically

significant. Given that after the first round of testing at

3 months post-injection, we killed five animals from each

of the four experimental groups; it is possible that our

experiment was insufficiently powered to detect milder

adverse effects arising from the different treatments. Sig-

nificant differences were also observed in the rearing fre-

quency in the staircase test. Namely, the Al-injected mice

showed a significantly lower frequency of rearing com-

pared to the group injected with Gardasil ? Pt in the

staircase test (p = 0.021; Fig. 1d). A lower frequency of

rearing is an indication of a reduced exploratory response

to a novel environment, and, it can also indicate a non-

selective attention deficit. There was no statistically sig-

nificant difference in the number of stairs climbed in the

staircase test between the groups (not shown). In the FST,

Environment and Autoimmunity

123

however, the changes were still significant despite the

lower number of animals. No significant differences in

behavior were observed in the novel object recognition test.

Autoantibody profile and inhibition assay

One month post-injection of either Al, Gardasil and Gar-

dasil ? Pt, the profile of serum antibodies was analyzed at

dilution of 1:200. Elevated levels of antibodies recognizing

the HPV L1 capsid protein of HPV types 6, 11, 16 and 18

(p\ 0.002), as well as anti-brain protein extract

(p\ 0.002) and anti-brain phospholipid extract antibodies

(p\ 0.001) were observed in the two groups of mice that

received the HPV vaccine (Fig. 2). The titers of anti-HPV

antibodies, anti-brain protein extract and anti-brain phos-

pholipid extract antibodies were reduced after 2 months

(data not shown). No elevation in the titers of anti-Al-

hydroxide, anti-dsDNA and anti-b2GPI antibodies, was

detected in the sera of any of the four treatment groups of

mice (Fig. 2).

The binding of anti-HPV antibodies from the sera from

the two treatment groups immunized with Gardasil to HPV

L1 antigens was significantly inhibited by the mouse brain

protein extract in a dose-dependent manner in comparison

with Al-injected mice whose sera were negative for anti-

HPV antibodies (Fig. 3).

Brain tissue immunostaining

Following the behavioral tests at 4.5 months of age, five

animals were killed from each of the four experimental

groups and used for brain immunostaining procedures.

With this relatively small group size, there were no clear

changes between the groups in both astrocyte and

Fig. 1 Effects of Al, Gardasil and Gardasil ? Pt toxin injections on

behavioral tests. a and b show the floating time in C57BL/6 female

mice as evaluated by the forced swimming test (FST). Results are

presented as duration in seconds (mean ± SEM) of immobility,

defined as the absence of escape-oriented behaviors, such as

swimming, jumping, rearing, sniffing or diving, recorded during the

6-min test. a Three months post-injection (n = 14 per treatment

group); b Six months post-injection (n = 9 per treatment group). b,c show the reduced exploratory activity in C57BL/6 female mice as

evaluated by the rearing frequency in the staircase test. Results are

presented as the number of rears (mean ± SEM) during a 3-min

testing period. a Three months post-injection (n = 14 per treatment

group); b Six months post-injection (n = 9 per treatment group)

Environment and Autoimmunity

123

microglia staining in any of the regions of interests we

investigated (CA1, CA3, dentate gyrus and the striatum).

Nonetheless, there was a significant difference between the

groups in the density of Iba-1 immunostaining using one-

tailed analysis (p = 0.046). Further post hoc analysis

revealed significant increase in Iba-1 density in the CA1 of

Gardasil-immunized mice compared to Al-injected mice

(p = 0.017; Fig. 4). These results suggest that the CA1

might be vulnerable to small changes in neuroinflammation

as a result of Gardasil immunization.

Discussion

The present results show alteration of behavioral responses

and neuro-inflammatory changes in mice as a result of Al

and Gardasil vaccine injection in exposure doses which are

equivalent to those in vaccinated human subjects. In par-

ticular, mice injected with Al and Gardasil spent signifi-

cantly more time floating in the FST test (measure

indicative either of locomotor dysfunction or depressive

behavior), compared to control animals (Fig. 1a, b). In

contrast, no significant differences were observed in the

number of stairs climbed in the staircase test which is a

measure of locomotor activity.

In addition, the Al-injected group showed abnormal

responses to a novel environment, which was manifested in

reduced rearing frequency in the staircase test, which

indicates a reduction in exploratory behavior (Fig. 1d). The

number of stairs and rears in this test is normally used to

provide measures of general physical motor abilities and

level of interest in the novelty of the environment. Rearing

in response to environmental change (i.e., removing a

mouse from the home cage and placing the animal in an

open box or a staircase apparatus) is also considered an

index of non-selective attention in rodents, while rearing

Fig. 2 Titers of serum antibodies 1 month post-injectionwith either Al

(A), Gardasil (G), Gardasil ? Pt toxin (Gp) and vehicle (V). A

homemade ELISA was used to detect the levels of anti-HPV, anti-Al

hydroxide (Alum), anti-mouse brain protein extract, anti-mouse brain

phospholipid (PL) extract, anti-dsDNA and anti-b2glycoprotein-I(b2GPI) antibodies in the sera of immunized mice. Pools of sera

(n = 5 per treatment group) were used as samples. All sera samples

were assayed in triplicate. Data are presented as mean OD 405 ± SEM

Fig. 3 Inhibition of the binding of antibodies from the sera of

Gardasil-injected mice to components of the vaccine (presumably the

HPV antigens) by the mouse protein extract. Pools of sera (n = 5 per

treatment group) were used as samples. All sera samples were assayed

by duplicates in independent experiments. Data are presented as mean

(% Inhibition) ± SEM where % inhibition = 100 - [(OD of tested

sample without inhibitor - OD of tested sample with inhibitor)/(OD

of tested sample without inhibitor)] 9 100 (inverted triangle) Al,

(filled balck circle) Gardasil, (open circle) Gardasil ? Pt

Fig. 4 Iba-1 immunostaining in the CA1 area of the hippocampus of

C57BL/6 female mice injected with Al, Gardasil and Gardasil ? Pt

toxin. Brain sections from five animals out of each group were

examined quantitatively for differences in immunostaining density

using Image J software (NIH, USA) as described in ‘‘Materials and

methods’’. The data are presented as % mean (% Intensity) ± SEM

Environment and Autoimmunity

123

during object investigation likely reflects selective atten-

tion [26].

We further observed significant increase in levels of

anti-HPV antibodies, and antibodies targeting the brain

protein and the brain phospholipid extract components in

the two groups of mice that received the Gardasil injection

(Fig. 2). Moreover, the recognition of vaccine components

(presumably the HPV L1 capsid protein species) by the

antibodies from the sera of Gardasil-immunized mice was

inhibited in a dose-dependent manner by the mouse brain

protein extract (Fig. 3). On the basis of these results, it

would appear that the anti-HPV antibodies from Gardasil-

vaccinated mice have the capacity to target not only the

HPV antigens but also brain antigen(s), either directly or

via negatively charged phospholipids. Finally, we observed

significant inflammatory changes in the Gardasil-injected

mice, namely the presence of activated microglia in the

CA1 area of the hippocampus (Fig. 4).

Possible mechanisms of vaccine-induced injury

The role of adjuvants

It is interesting to note that, in our hands, the extent of

adverse neurological manifestations was similar in the

three treatment groups whose only common denominator

was the Al compound. As we noted above, the clinical

trials for both HPV vaccines, Gardasil and Cervarix, used

an Al-containing placebo and the safety of the vaccines

was thus presumed on the finding that there was an equal

number of adverse events in the vaccine and the alleged

placebo group [21, 22, 27–31]. The HPV vaccines, like

many other vaccines, are adjuvanted with Al in spite of

well-documented evidence that Al can be both neuro- and

immuno-toxic [10, 11, 13, 32–35] and hence does not

constitute an appropriate placebo choice.

The appearance of diverse adverse neurological and

immuno-inflammatory manifestations following routine

vaccinations is well documented in the medical literature

(Table 1). Although the classical explanations for these

phenomena have largely centered on vaccine antigens, in

recent years attention has shifted to Al adjuvants. Conse-

quently, in the last decade, studies on animal models and

humans have indicated that Al adjuvants have an intrinsic

ability to inflict adverse immune and neuro-inflammatory

responses [9–11, 13, 14, 33, 35–37]. This research culmi-

nated in delineation of ASIA-‘autoimmune/inflammatory

syndrome induced by adjuvants’, which encompasses the

wide spectrum of adjuvant-triggered medical conditions

characterized by a misregulated immune response [2].

Notably, the vast majority of adverse manifestations

experimentally triggered by Al in animal models and those

associated with administration of adjuvanted vaccines in

humans are neurological and neuropsychiatric [2]. These

observations should not be particularly surprising given

Al’s well-established neurotoxic properties [38, 39]. What

has, however, been argued is that the concentrations at

which Al is used in vaccines are not sufficient to cause

neurotoxicity [17, 40]. This argument, however, is not

supported by recent evidence.

It should be noted that the long-term biodistribution of

nanomaterials used in medicine is largely unknown. This is

likewise the case with the Al vaccine adjuvant, which is a

nanocrystalline compound spontaneously forming

micron/submicron-sized agglomerates. It has been recently

demonstrated that Al adjuvant compounds from vaccines,

as well as Al-surrogate fluorescent nanomaterials, have a

unique capacity to cross the blood–brain and blood–cere-

brospinal fluid barriers and incite deleterious immuno-in-

flammatory responses in neural tissues [10, 13, 41]. Thus, a

proportion of Al particles escapes the injected muscle,

mainly within immune cells, travels to regional draining

lymph nodes, then exits the lymphatic system to reach the

bloodstream eventually gaining access to distant organs,

including the spleen and the brain. Moreover, the Trojan

horse mechanism by which Al loaded in macrophages

enters the brain, results in the slow accumulation of this

metal, due to lack of recirculation [10, 41]. The sustained

presence of Al in central nervous system tissues is likely

responsible for the myriad of cognitive deficits associated

with administration of Al-containing vaccines in patients

suffering from post-vaccination chronic systemic disease

syndromes including macrophagic myofasciitis (MMF)

[9, 11, 35].

Thus, contrary to prevalent assumptions, Al in the

adjuvant form is not rapidly excreted but rather, tends to

persist in the body long-term. As demonstrated by Khan

et al. [41], intramuscular injection of Al-containing vaccine

in mice is associated with the appearance of Al deposits in

distant organs, such as spleen and brain, which were still

detected 1 year after injection. Similarly, Al-particle fluo-

rescent surrogate nanomaterials injected into muscle were

found to translocate to draining lymph nodes and thereafter

were detected associated with phagocytes in blood and

spleen. Particles linearly accumulated in the brain up to the

6-month end point. They were first found in perivascular

CD11b ? cells and then in microglia and other neural

cells. The ablation of draining lymph nodes dramatically

reduced the biodistribution of injected Al-fluorescent sur-

rogate nanocompounds. In addition, the nanoparticle

delivery into the brain was found to be critically dependent

on the major monocyte chemoattractant protein MCP-1/

CCL2 as intramuscular injection of murine rCCL2 strongly

increased particle incorporation into intact brain while

CCL2-deficient mice had decreased neurodelivery [41].

Environment and Autoimmunity

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In the ASIA syndrome, there could be a the prolonged

hyperactivation of the immune system and chronic

inflammation triggered by repeated exposure and unex-

pectedly long persistence of Al adjuvants in the human

body (up to years post-vaccination) [6, 42]. It is probable

that one of the reasons why Al adjuvants are retained long-

term in bodily compartments including systemic circula-

tion is due to their tight association with vaccine antigens

or other vaccine excipients [43]. Even dietary Al has been

shown to accumulate in the central nervous system over-

time, producing Alzheimer’s disease type outcomes in

experimental animals given dietary equivalent amounts of

Al to what humans consume through a typical Western diet

[44].

The ability of Al adjuvant nanoparticles to cross the

blood–brain barrier via a macrophage-dependent Trojan

horse mechanism may explain in part why some vaccines

have a predilection to affect the central nervous system

[8, 10, 33, 35, 39]. Another explanation comes from the

fact that Al nanomaterials can on their own damage the

blood–brain barrier and induce neurovascular injury

[16, 45]. Collectively, these studies [16, 41, 45] show that

nano-Al can accumulate in brain cells, inducing nerve and

blood vessel damage and protein degradation in the brain.

Persistent accumulation of nano-Al compounds regardless

the source (i.e., vaccines, dietary) in the central nervous

system may thus increase the likelihood of the develop-

ment of acute and/or chronic neurological disorders.

With respect to the particular Al compounds used in

HPV vaccines, AAHS in Gardasil and ASO4 (3-0-desacyl-

40-monophosphoryl lipid A (MPL) adsorbed onto Al

hydroxide) in Cervarix, it should be noted that these new

adjuvants induce a much stronger immune response than

conventional Al adjuvants used in other vaccines (i.e., Al

hydroxide and Al phosphate) [46]. Stronger immuno-

genicity of an adjuvant formulation also implies by default

stronger reactogenicity and risk of adverse reactions.

Because of the differences in immune-stimulating proper-

ties between different Al adjuvant compounds, safety of a

particular adjuvant formulation cannot be a priori assumed

on the basis of the allegedly good historical track record of

other formulations. Rather, they need to be thoroughly

evaluated case by case.

According to the US FDA, a placebo is, ‘an inactive pill,

liquid, or powder that has no treatment value’ [47]. From

the literature cited above as well as the present study, it is

obvious that Al in adjuvant form is neither inactive nor

harmless and hence cannot constitute as a valid placebo.

Commenting on the routine practice of using Al-based

adjuvants as placebos in vaccine trials Exley recently stated

that it is necessary to make a very strong scientific case for

using a placebo which is itself known to result in side

effects and that no scientific vindication for such practice is

found in the relevant human vaccination literature [7].

Conceivably, there is even less justification for using a

novel and more potent Al formulation than those that have

been in standard use (Al phosphate and hydroxide). The

only aim that this practice achieves is to give potentially

misleading data on vaccine safety. Moreover, it is unethical

to give a placebo to healthy clinical trial subjects that has

no benefit but rather, may cause harm.

The role of vaccine-induced antigens: immune cross-

reaction

As noted above, we observed significant elevation of

antibodies recognizing Gardasil components, most likely

the HPV L1 capsid protein of HPV types 6, 11, 16 and 18

(p\ 0.002) and of antibodies targeting the mouse brain

protein (p\ 0.002) and phospholipid extracts (p\ 0.001)

in the sera of Gardasil-immunized mice (Fig. 2). The

binding of anti-HPV antibodies from the sera of mice

injected with Gardasil to components of the HPV vaccine,

presumably the HPV L1 antigens, was inhibited in a dose-

dependent manner by using mouse brain protein extract as

the inhibitor (Fig. 3). Taken together, these results suggest

that antibodies from Gardasil-vaccinated mice have the

capacity to target not only the HPV L1 antigens but also

brain antigen(s), either directly or via negatively charged

phospholipids.

This interpretation is consistent with the findings of

Kanduc [48] who showed that antigen present in both HPV

vaccines Gardasil and Cervarix (the major capsid L1 pro-

tein of HPV-16) shares amino acid sequence similarity

with numerous human proteins, including cardiac and

neuronal antigens, human cell-adhesion molecules,

enzymes and transcription factors. Moreover, such con-

tention is also supported by a case of severe acute cere-

bellar ataxia (ACA) following HPV vaccination where

combined immunosuppressive therapy with methylpred-

nisolone pulse and intravenous immunoglobulin (IVIG)

therapies as well as immunoadsorption plasmapheresis

resulted in complete recovery of the patient. In this par-

ticular case, the patient (12-year-old girl) developed

symptoms of ACA, including nausea, vertigo, severe limb

and truncal ataxia, and bilateral spontaneous continuous

horizontal nystagmus with irregular rhythm, 12 days after

administration of the HPV vaccine. Severe ACA symptoms

did not improve after methylprednisolone pulse and IVIG

therapies, but the patient recovered completely after

immunoadsorption plasmapheresis [49]. Although no sig-

nificant antibodies were detected in this patient, the

remarkable effectiveness of immunoadsorption plasma-

pheresis strongly suggested that some unidentified anti-

bodies were involved in the pathophysiology of ACA [49].

Citing the work of Kanduc [50], the authors of this case

Environment and Autoimmunity

123

have stated that further research on molecular mimicry

between human proteins and HPV16 L1-derived peptide is

needed to determine the exact pathologic mechanism of

ACA [49]. Altogether, these observations suggest that

possible immune cross-reactions derived from utilization

of HPV L1 antigens in current HPV vaccines might be a

risk for cardiovascular and neurological autoimmune

abnormalities [48, 50]. Our observation that nearly 85 %

(129/152) of HPV vaccine adverse case reports in the

current scientific literature relate to neuro-ophthalmic

abnormalities may lend further support for this conclusion

(Table 1).

Conclusions

In summary, both Al and Gardasil vaccine injections

resulted in behavioral abnormalities in mice (Figs. 1, 2, 3).

Furthermore, immunostaining analysis showed an increase

in the Iba-1 density in the CA1 area of the hippocampus in

Gardasil-immunized mice in comparison with Al-injected

mice, thus suggesting that CA1 might be vulnerable to

neuroinflammation as a result of Gardasil immunization

(Fig. 4).

In addition, we observed that the brain protein extract

significantly inhibited in a dose-dependent manner, the

binding of total IgG isolated from the sera of Gardasil-

immunized mice to components of the vaccine, most likely,

the HPV L1 capsid antigenic component (Fig. 3). There-

fore, it is likely that mice immunized with the HPV vaccine

developed cross-reactive anti-HPV antibodies which in

addition to binding to the HPV L1 capsid protein may also

bind to brain auto-antigens. The putative target anti-

gen(s) should be further identified by immunoprecipitation

and proteomics analyses.

In light of these findings, this study highlights the

necessity of proceeding with caution with respect to further

mass-immunization practices with a vaccine of yet unpro-

ven long-term clinical benefit in cervical cancer prevention

[20, 51] and which in the other hand is capable of inducing

immune-mediated cross-reactions with neural antigens of

the human host. This note of caution becomes even more

relevant when considering the continually increasing

number of serious disabling neurological adverse events

linked to HPV vaccination reported in the current medical

literature (Table 1) and in vaccine surveillance databases

[20].

Finally, in light of the data presented in this manuscript,

new guidelines should be requested on the use of appro-

priate placebos in vaccine safety trials [7].

Compliance with ethical standards

Conflict of interest Yehuda Shoenfeld has acted as a consultant for

the no-fault US National Vaccine Injury Compensation Program. L.T.

has served as an expert witness in cases involving adverse reactions

following qHPV vaccine administration. The other co-authors declare

no competing interests.

Funding This work was supported by the grants from the Dwoskin

Foundation Ltd.

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